The invention is in the field of devices and methods for sequencing biopolymers.
The prevailing DNA sequencing methods are based on Sanger chemistry and on fragment analysis using gel-based electrophoresis. These methods reveal the base pair sequence of individual fragments of DNA. The bases are separated by subjecting the fragments, suspended in a slab gel, to an electrical field. This causes size-dependent migration and spatial separation of the fragments. Once they have been separated on the gel, the bands corresponding to the individual base pairs are read or digitally scanned to determine the fragment sequence.
Although the results obtained from gel electrophoresis are generally of high quality and reliability, the process is labor intensive and relatively slow. Due to its complexity, gel electrophoresis often requires a skilled technician. Additionally, preparing samples prior to sequencing requires that the target be isolated, purified, amplified, and fragmented into relatively smaller pieces (e.g., about 300 to 500 base pairs). Since the average length of a human gene is over 50,000 bases (i.e., 15,000 to over 1,000,000 bases), considerable sample preparation is necessary to systematically fragment, purify, and amplify the fragments.
To ensure that each fragment is sequenced at least once, the target section is often deliberately overlapped, with the consequence that the same bases may be sequenced ten times or more in the end. Once the sequencing has been completed, the resulting data are processed to deduce the sequence of the original target section.
Improved engineering and automation have resulted in sequencing systems that include such technological advances as automated gel-based electrophoresis or ultra-thin capillary tube electrophoresis. These techniques permit higher speed and lower cost sequencing but are still limited by the fundamental constraints of Sanger-based chemistry and fragment analysis, namely the need for highly trained personnel to prepare relatively short read lengths. Nonetheless, automated gel electrophoresis is the technique currently used for almost all high throughput commercial sequencing. Current efforts toward gene discovery, for example those based on "population-based" genetic analysis, can create tremendous demand for cost-effective DNA sequence analysis. For example, HIV protease inhibitor medications recently introduced rely heavily on DNA sequencing of individual patient samples to detect the emergence of resistant strains of HIV and subsequently alter choice of therapeutic intervention. Indeed, cost-effective DNA sequence analysis methods are likely to prove to be a prerequisite for "individual-based" preclinical and clinical patient studies.